Uranium (U) is the forty-ninth most abundant element in the Earth's crust. While there are 17 known isotopes of U, it occurs in the environment primarily as 3 of those known isotopes, namely 238U (99.27%), 235U (0.72%), and 234U (0.005%). Although, the known isotopes of U are radioactive, the greatest ecological and/or environmental concerns are raised by their chemical toxicity rather than radioactivity. Anthropogenic use of U has led to environmental contamination. And, the disposal of electronic devices such as cell phones and computers can contribute to potential environmental contamination as some of these electronic components contain heavy metals, lead, mercury, selenium, cadmium, and hafnium. These types of contaminants may leach from waste sites and cause environmental contamination. Thus, there is a need to dispose of U, as well as other radionuclides, for example, plutonium and thorium and toxic metals such as cobalt, chromium, copper, lead, zinc, nickel and manganese. In order to return a waste site back to a useful condition, it must be decontaminated by removing both the metal and the radionuclide contaminants from the contaminated site. Stabilizing and reducing the mass of radionuclides and toxic metals contained in contaminated materials would facilitate their disposal. As used herein, stabilizing means the treatment of radionuclides and toxic metals so that they are in a stable or insoluble form and lack the ability to be easily converted chemically or biologically to another soluble form. One such method of treatment is bioremediation.
Bioremediation of metals and radionuclides, including U, is distinctly different than biodegradation of toxic organic substances because toxic metals, for example, cannot be degraded. Thus, bioremediation of toxic metals and radionuclides depends on a method of containment that decreases their bioavailability and/or biological access. It is well known that microorganisms can undergo processes that transform and transport radionuclides and toxic metals. Essentially, radionuclides and toxic metals in waste, that are present in soluble form, can be converted into an insoluble form by chemical or microbiological processes. Examples of such microbiological reactions include oxidation/reduction, complexation, change in pH and Eh which affect the valence state of the metal as well as its solubility characteristics, production of sequestering agents, and bioaccumulation. Each of these processes can lead to attenuation or mobilization of metals in the environment.
Bioremediation of U-contaminated waste has been studied over the past 15 years largely because of its chemistry. The oxidation state of U is crucial to its stability, mobility and bioavailability. The oxidized or hexavalent, (VI), state of U is highly soluble and therefore, mobile, while the reduced or tetravalent, (IV), state is relatively insoluble. In waste, U is present primarily as soluble salts of the uranyl ion (UO22+) When the uranyl ion is reduced from the U(VI) oxidation state to a lower oxidation state such as U(IV), the solubility decreases and it becomes immobilized. A number of bacteria is known to reduce U(VI) and include, for example, Cellulomonas sp., Clostridium sp., Desulfosporosinus spp. Desulfovibrio sp., as well as others. Although there is still not a complete understanding of the biochemistry of the process involved using any one bacterium, it has been reported that a common factor among all of the bacteria known to reduce U(VI) is the ability to grow anaerobically where a redox potential sufficiently low for U(VI) reduction would be established.
Microbial reduction of U(VI) was first reported in crude extracts from Micrococcus lactilyticus (reclassified as Veillonella alcalescens) by assaying the consumption of hydrogen dependent on the presence of U(VI). It was generally believed that abiotic processes were responsible for the production of U(IV) in anaerobic or low redox environments, by processes that included reduction by sulfide, Fe(II), or hydrogen. It was established afterwards that microbial U(IV) reduction was achieved by dissimilatory metal-reducing bacteria by assaying with pure cultures of the Fe(III)-reducing bacteria, Geobacter metallireducens strain GS-15 and Alteromonas putrefaciens (later Shewanella putrefaciens). Conversion of U(VI) to insoluble U(IV) was followed as a decrease in absorption of U(VI) at 424.2 nm with a directly coupled plasma spectrometer after separation of the two U forms by ion exchange chromatography. It was also shown that live cells and an oxidizable substrate were necessary for the U(VI) transformation.
Reductive precipitation of U using microorganisms is presently accepted as a potential strategy for removal of soluble radionuclides and toxic metals to prevent their further migration. In general, current technologies work by stimulation or inoculation of a microorganism directly into the material to be treated. In another method, absorption of contaminant material on to bacterial surfaces (biosorption) has been used. For example, a biological method for removing U from waste (e.g., water), involves adding glycerol-2-phosphate to the U-containing water and then treating the waste water with the microorganism Citrobacter sp. This microorganism is reported to have a phosphatase enzyme that releases phosphate from the glycerol-2-phosphate which then forms an insoluble uranium precipitate on the cell surface of the bacteria (biosorption). The enzymatic reaction does not involve uranium and disadvantages to this process are that it is hindered by the presence of carbonate; it precipitates metals, other than U, that form an insoluble phosphate complex; the amount of U that can be sorbed onto the cell surface is limited; and the sorbed U represents a large volume of waste that contains only a small fraction of U and a large quantity of bacterial biomass.
Another conventional method for bioremediation of U is by contacting an iron oxide-metal coprecipitate with a bacterial culture containing Clostridium sp. ATCC No. 53464 in a nutrient medium which satisfies the nutritional requirements of the bacterial culture. The treatment of the coprecipitate with the culture of Clostridium sp. or its metabolites, solubilizes the ions. The bacterial culture is added to or inoculated or stimulated in the contaminated sludge/water. However, the disadvantage to this method is that the radionuclides and toxic (heavy) metals, because they are toxic to the microbes, have a limited concentration for removal. The microbe can only tolerate up to a critical concentration of about 0.2 mM of U, above which its effect (between about 0.21 mM and 0.42 mM of U) in stabilizing the ions diminishes because concentrations greater than about 0.2 mM are known to be toxic to the bacteria, Clostridium sp. It has been reported that a concentration of about 0.42 mM of U is not treated by the bacteria, Clostridium sp.
Further it has been reported that S. oneidensis strain MR-1, Shewanella species (S. alga strain BrY), S. putrefaciens strain 200 and Pseudomonas stutzeri strain demonstrate capabilities in the production of microbially based electron shuttles for electron transfer. However, these strains are gram negative bacteria which have thin cell wall layers, and therefore, it is not surprising that they have those capabilities. Thus, there remains a need to further expand and enhance the methods of bioremediation. To this end, the present invention relates to a method of bioremediation capable of using microorganisms for treating toxic metals.